Our final goal is to achieve targeted integration of large (>25kb) transgene cassettes in human hematopoietic stem cells (HSCs). Towards to goal, we constructed helper-dependent, fiber-chimeric adenovirus vectors (HD-Ad5/35) that transduce HSCs. HD-Ad5/35 vectors are very efficient in delivering large transgene cassettes into the nucleus of HSCs where the vector genomes remain in an episomal stage, packaged into nucleosome-like structures. However, when HD-Ad5/35 vectors contained a 23kb fragment of the 2-globin locus control region (HD-Ad5/35.LCR-1), vector genomes integrated at a high frequency into the chromosomal DNA of human erythroid Mo7e cells. Importantly, 20% of all integration events occurred within the chromosomal 2-globin LCR, particularly into a region within and downstream of hypersensitivity site 2. We demonstrated that targeted HD-Ad5/35.LCR-1 integration involves globin LCR-specific proteins characteristic for "active" chromatin and that incoming HD-Ad5/35.LCR-1 genomes are physically tethered to the chromosomal globin LCR involving chromatin proteins. We speculate that physical proximity between vector and chromosomal DNA, together with DNA breaks within the vicinity of tethered vector DNA, mediates preferential integration of our vector into the globin LCR in Mo7e cells. In this proposal we will attempt to achieve targeted transgene integration in HSCs. In preliminary studies, LCR-chromatin tethering and/or vector integration is less efficient in CD34+ cells than in Mo7e cells. We speculate that the 2-globin LCR chromatin in CD34+ cells is competent for HD-Ad5/35.LCR-1 tethering and the limiting step in stable CD34+ cell transduction is vector integration. We will test this hypothesis in Specific Aim 1. Our Specific Aim 2 is to improve the integration efficiency of tethered HD-Ad5/35.LCR genomes using a &C31-phage integrase. Overall, this work will lead us to a better understanding of mechanisms and structural determinants of chromatin tethering and its role in facilitating vector integration and create the basis for improving targeted integration of existing retrovirus vectors through chromatin tethering of retroviral integrases. PUBLIC HEALTH RELEVANCE: We propose a novel vector system to achieve targeted integration of a large transgene cassette in human hematopoietic stem cells. Our approach combines a new vehicle for DNA delivery into stem cells with a new idea to achieve targeted integration of a large transgene cassette. Specifically, we plan to capitalize on our recent finding that preferential vector integration into the beta-globin LCR can be achieve via chromatin tethering of transgene cassettes. This study is relevant for engineering target site specificity of integrating vectors in general and might provide a basis for globin gene therapy.